Japanese Cedar Pollen Allergens in Japan.

Pollen from members of the Cupressaceae tree family is one of the most important causes of allergic disease in the world. Cryptomeria japonica (Japanese cedar) and Chamaecyparis obtusa (Japanese cypress) are Japan's most common tree species. The pollen dispersal season is mainly from February to May. The major allergens of Japanese cedar and Japanese cypress exhibit high amino acid sequence similarity due to the phylogenetic relationship between the two species. An epidemiological study has shown that the prevalence of Japanese cedar pollinosis is approximately 40%. Younger children (5 to 9 years old) showed a high prevalence of Japanese cedar pollinosis as 30% in 2019, indicating that season pollinosis is getting worse. Pharmacotherapy is the most common treatment for pollinosis induced by Japanese cedar and Japanese cypress. Patients' satisfaction with pharmacotherapy is low due to insufficient experienced effect and daytime somnolence. Unlike pharmacotherapy, allergy immunotherapy (AIT) addresses the basic immunological mechanisms of allergic disease and activates protective allergen-reactive pathways of the immune system. AIT is now recognized as the only treatment option with the potential to provide long-term post-treatment benefits and alter the natural course of the allergic disease, including Japanese cedar pollinosis.

Correlation between sensitization to house dust mite major allergens, age, and symptoms in Japanese house dust mite allergic subjects.

Der p 23 has recently been recognized as a new house dust mite (HDM) major allergen that may be linked to the development of asthma in HDM allergic patients. This study aimed to investigate the frequency of sensitization to HDM major allergen components including Der p 23 and to examine the correlation between HDM-sensitization and AR symptom score in Japanese HDM allergic rhinitis (AR) patients without allergic asthma. Serum samples (n = 120) collected from Japanese HDM AR patients (12 to 64 years) without asthma were assessed for allergen-specific IgE (s-IgE) by ImmunoCAP (Dermatophagoides pteronyssinus (D. pteronyssinus; Der p) extract, Der p 23) or immunosolid-phase allergen chip (Der p 1, Der p 2). Japanese HDM AR patients without asthma showed a high prevalence of allergic sensitization to the HDM major allergens Der p 1 (94.2%), Der p 2 (97.5%) and Der p 23 (71.7%). No difference in the prevalence was detected for Der p 1 and Der p 2 s-IgE among three age groups. However, the prevalence of Der p 23 s-IgE was significantly higher in the younger group compared to the elderly group. No significant correlation was found between AR symptom scores and concentration of s-IgE towards Der p extract and any of the three HDM major allergens. Although the prevalence of sensitization towards D. pteronyssinus major allergens is high in Japanese AR patients without asthma, there was no correlation between allergen specific IgE including IgE towards Der p 23 and AR symptom in this population.

Reduced anaphylactic potential of denatured pullulan-conjugated Cry j 1.

Japanese Cedar (JC) pollinosis is the most common seasonal allergic rhinitis in Japan. Throughout the JC pollen season, patients suffer from the allergic symptoms, resulting in a reduction of quality of life. Allergy immunotherapy (AIT) is an established treatment option for a wide range of allergens that unlike symptomatic treatments (e.g. antihistamines) may provide sustained immune tolerance. However, AIT, especially subcutaneous immunotherapy (SCIT) has a fatal anaphylaxis risk due to the use of crude allergen extracts. Consequently, development of allergen derivatives with substantially reduced anaphylactic potential is desirable. An allergen derivative that showed reduced IgE-binding and anaphylactic potential was developed through conjugation of native Cry j 1 (n Cry j 1), a major JC allergen, to the polysaccharide pullulan followed by chemical but non-covalent denaturation. The resulting Cry j 1 allergen derivative, Dn p-Cry j 1, showed reduced IgE-binding and IgE-mediated effector cell activation in vitro using an ELISA competition assay and a mast cell activation model (EXiLE). Reduced anaphylactic potential of Dn p-Cry j 1 in vivo was demonstrated using the rat passive cutaneous anaphylaxis (PCA) assay. The difference in anaphylactic potential of Dn p-Cry j 1 compared to n Cry j 1 in wild-type rats was of the same magnitude as the difference seen in the anaphylaxis reactions obtained with n Cry j 1 in wild-type rats and mast-cell deficient rats, indicating a dramatic reduction in anaphylactic potential of Dn p-Cry j 1. These results indicate that Dn p-Cry j 1 is a promising candidate for next-generation JC AIT.

Latent 1,3-ß-D-glucan acts as an adjuvant for allergen-specific IgE production induced by Japanese cedar pollen exposure.

BACKGROUND: The pollen grains of several plant species contain 1,3-ß-D-glucan (BG). BG activates dendritic cells (DCs) and subsequently regulates the innate immune responses. Within Japan, the most common disease associated with type-I hypersensitivity is Japanese cedar pollinosis. However, the role of BG in Japanese cedar pollen (JCP) remains unclear. This study examined the localization and immunological effects of BG in JCP. METHODS: The localization of BG in JCP grain was determined by immunohistochemical staining using a soluble dectin-1 protein probe and a BG recognition protein (BGRP). The content of BG extracted from JCP was measured by a BGRP-based ELISA-like assay. The cytokine production by bone marrow-derived DCs (BMDCs) obtained from wild-type and BG receptor (dectin-1) knock-out mice was examined in vitro. The mice were intranasally administered JCP grains and the specific serum Ig levels were then quantified. RESULTS: BG was detected in the exine and cell wall of the generative cell and tube cell of the JCP grain. Moreover, BG in the exine stimulated production of TNF-α and IL-6 in the BMDCs via a dectin-1-dependent mechanism. Meanwhile, JCP-specific IgE and IgG were detected in the serum of wild-type mice that had been intranasally administered with JCP grains. These mice also exhibited significantly enhanced sneezing behavior. However, dectin-1 knock-out mice exhibited significantly lower JCP-specific IgE and IgG levels compared to wild-type mice. CONCLUSIONS: Latent BG in JCP can act as an adjuvant to induce JCP-specific antibody production via dectin-1.

Formation of IgE-Allergen-CD23 Complex Changes in Children Treated with Subcutaneous Immunotherapy for Japanese Cedar Pollinosis.

BACKGROUND: Subcutaneous immunotherapy (SCIT) is used to treat Japanese cedar (JC) pollinosis. The formation of IgE-allergen-CD23 complex after SCIT for JC pollinosis has not yet been fully elucidated. OBJECTIVE: The objective of this study was to investigate the formation of IgE-allergen-CD23 complex after SCIT for JC pollinosis. METHODS: Eleven patients were treated with 3-year SCIT for JC pollinosis at Sa-gamihara National Hospital from 2013 to 2014. Nasal and ocular symptoms (in terms of symptom scores) during the scattering of JC pollen and immunological changes were investigated. Levels of JC pollen-specific antibodies (IgE and IgG4) were measured by ImmunoCAP assays. To detect the changes in allergen-presenting ability of B cells, the levels of IgE-allergen-CD23 complexes in serum were measured by a cell-free, enzyme-linked immunosorbent-facilitated antigen-binding assay. RESULTS: The median (interquartile range) age of the subjects was 8 (6-10) years. Three patients (27%) had comorbid atopic dermatitis, and 5 patients (45%) had comorbid bronchial asthma. Before starting SCIT, the total IgE level was 373 (75-2,870) kU/L, and the level of JC pollen-specific IgE was 77.2 (15.4-528) kUA/L. Symptom scores improved significantly from the year after treatment. JC pollen-specific IgE levels did not change after 3 years of treatment. JC pollen-specific IgG4 levels increased significantly throughout the treatment period. The levels of IgE-allergen-CD23 complexes decreased significantly after 3 years of treatment. CONCLUSION: The ability of IgE-allergen complexes to bind to CD23 decreased after SCIT, suggesting that increasing levels of IgE-blocking antibodies, including IgG4, may play an important role in the mechanism of SCIT.

Allergen Stability and Immunological Reactivity during Co-dissolution and Incubation of House Dust Mite and Japanese Cedar Pollen SLIT-Tablets.

Japanese allergic subjects are commonly sensitized to both house dust mite (HDM) and Japanese cedar pollen (JCP) and combined treatment with sublingual immunotherapy (SLIT) tablets is desirable. However, mixing extracts of two non-homologous allergens may compromise allergen stability and affect the clinical outcome. Therefore, we investigated the stability of major allergens and total allergenic reactivity of HDM and JCP SLIT-tablets following dissolution in human saliva or artificial gastric juice. Two fast-dissolving freeze-dried SLIT-tablets were completely dissolved and incubated at 37 °C. Major allergen concentrations and total allergenic reactivity were measured. After mixing and co-incubation of HDM and JCP SLIT tablets in human saliva for 10 min at 37°C, there were no statistically significant changes in major allergen concentrations. In addition, no loss of allergenic reactivity of the mixed two SLIT-tablet solutions was seen. In contrast, complete loss of allergenic reactivity and detectable major allergen concentrations occurred when the two SLIT-tablets were dissolved and incubated in artificial gastric juice. These results demonstrate that HDM or JCP major allergens and the total allergenic reactivity of both SLIT-tablets measured here remain intact after dissolution and co-incubation in human saliva, supporting the possibility of a dual HDM and JCP SLIT-tablet administration regimen if clinically indicated. The complete loss of allergenic reactivity after incubation in artificial gastric juice can furthermore be taken to indicate that the immunological activity of the allergen extracts contained in the two SLIT-tablets is likely to be lost or severely compromised upon swallowing.

Establishment of enzyme-linked immunosorbent facilitated antigen binding as a biomarker assay for Japanese cedar pollen allergy immunotherapy.

BACKGROUND: Clinical efficacy of allergen-specific Immunotherapy (AIT) towards Japanese cedar (JC) pollen allergy is firmly established but JC pollen-specific biomarker assays are lacking. Treatment-related increase of allergen-specific antibodies is a robust biomarker of successful AIT. Allergen-specific non-IgE antibodies are believed to reduce the effects of allergen exposure by competing with IgE for allergen binding, and in-vitro assays quantifying the effects of AIT-induced IgE-blocking antibodies are advantageous. A cell-free enzyme-linked immunosorbent facilitated antigen binding (ELIFAB) assay of JC pollen was established. METHODS: Serum IgE-allergen complexes were captured by immobilized recombinant CD23, and allergen-IgE-CD23 complexes were detected by a biotin-conjugated anti-human IgE antibody. Sera from JC pollen-allergic subjects without or with subcutaneous immunotherapy (SCIT) with JC pollen extract were used (n = 11/group). RESULTS: Optimal assay conditions were established at 20 µg/mL CD23 and 0.3 µg/mL JC pollen extract, and the dependency on CD23 and IgE was verified. The data show that the JC pollen ELIFAB assay is fit for purpose and demonstrates that the IgE-blocking activity is significantly increased in the JC pollen SCIT group compared with the non-treated group. CONCLUSION: The JC pollen ELIFAB assay represents a simple, cell-free biomarker assay for monitoring the development of IgE-blocking antibody activity during JC pollen AIT.

The Importance of Tablet Formulation on Allergen Release Kinetics and Efficiency: Comparison of Freeze-dried and Compressed Grass Pollen Sublingual Allergy Immunotherapy Tablet Formulations.

PURPOSE: Efficient delivery of allergens to the sublingual mucosa is a prerequisite for successful sublingual immunotherapy (SLIT) for allergy, and in order to become available to immune-competent cells embedded in the sublingual mucosa, allergens need to be delivered in a soluble form. Delivery of solubilized allergens poses a particular challenge for tablet-based allergy immunotherapy, in which allergens are administered under the tongue in the form of dry tablets and need to be dissolved rapidly in a small volume of saliva, with little or no agitation. The purposes of this article were to compare the properties of 2 different pharmaceutical SLIT-tablet formulations, freeze-dried and compressed, and to examine how the tablet formulation affects the efficiency with which allergen is delivered from the dry state of the tablet into soluble form. METHODS: Two SLIT-tablet formulations, both indicated for grass pollen allergic rhinitis and containing grass pollen extract as the active ingredient, were examined with regard to tablet disintegration times, allergen dissolution kinetics, dependency on solvent volume and agitation, and the achieved recovery of the grass allergen content in soluble form with each tablet. FINDINGS: The freeze-dried and the compressed SLIT-tablet formulations differed markedly with respect to efficiency of allergen release. The freeze-dried tablet disintegrated faster and released grass allergen into solution with a release rate higher than that of the compressed formulation and, in contrast to the compressed formulation, achieved full recovery of the allergen content in soluble form in a small volume of solvent. IMPLICATIONS: Rapid and complete release of soluble allergen in a small volume of solvent, as demonstrated by the freeze-dried formulation, are key elements of efficient sublingual allergen delivery by SLIT-tablets. Complete allergen release means that the full allergen dose of the tablet is recovered from the tablet and made available to the sublingual immune system in soluble form, and rapid release ensures that the immune system becomes exposed to the highest possible dose of soluble allergen for the maximal duration before swallowing. In contrast, a SLIT-tablet formulation that provides incomplete and slower allergen release will likely require a higher allergen content compared to the more efficient formulation, in order to achieve the same dose of soluble allergen, consequently leading to an excess load of allergen that becomes swallowed without having been made immunologically available.

Safety evaluation of standardized allergen extract of Japanese cedar pollen for sublingual immunotherapy.

Japanese cedar (JC) pollinosis is caused by Japanese cedar pollen (JCP) and most common seasonal allergic disease in Japan. Subcutaneous immunotherapy (SCIT) with allergen extract of JCP (JCP-allergen extract) is well established for JC pollinosis treatment with improvement of symptoms. However, major drawbacks for SCIT are repeated painful injections, frequent hospital visits and anaphylactic risk. Currently, sublingual immunotherapy (SLIT) has received much attention as an advanced alternative application with lower incidence of systemic reactions because the liquid or tablet form of allergen is placed under the tongue. The aim of this study was safety evaluation of standardized JCP-allergen extract currently developed for SLIT in JC pollinosis. JCP-allergen extract showed no potential genotoxicity. No systemic effects were observed in rats administered JCP-allergen extract orally for 26 weeks followed by 4-week recovery period. Mild local reactions such as hyperplasia and increased globule leukocytes resulting from vehicle (glycerin)-induced irritation were observed in stomach. No-observed-adverse-effect level was greater than 10,000 JAU/kg/day for systemic toxicity, equivalent to 300-fold the human dose. No local irritation was found in rabbits oral mucosae by 7-day sublingual administration. These results demonstrate the safe profile of standardized JCP-allergen extract, suggesting it is suitable for SLIT in JC pollinosis.